Improved Spectrophotometric Determination of Bismuth ( III ) with Bromopyrogallol Red in Mixed Surfactants-Application to Waters and Veterinary Preparation

A simple and sensitive spectrophotometric method for the determination of trace amounts of bismuth is established. The method is based on the reaction of bismuth with bromopyrogallol red at pH 3 in the presence of a mixture of cetylpyridinium chloride and triton X-100 surfactants to form a blue coloured complex which shows maximum absorbance at 644 nm. A plot of absorbance with bismuth concentration gives a straight line indicating that Beers law has been obeyed over the range 3-150 μg/25 ml, i.e., 0.12-6 ppm with a molar absorptivity of 3.3×10 l.mol.cm and Sandells sensitivity index of 0.006 μg.cm. The interfering effect of a number of common captions and anions in the presence of comosite mixture (NaF and tartaric acid) and ascorbic acid as masking agents has been studied. The effect of common excipients has also been examined. The method has been applied successfully to the determination of bismuth in water samples and a veterinary preparation.

The present work is devoted to the spectrophotometric study of the coloured complex of bismuth with bromopyrogallolred in the presence of mixture of cetylpyridinum chloride and Triton X-100 surfactants as an attempt to increase the sensitivity and selectivity of bismuth determination.

EXPERIMENTAL
Spectral and absorbance measurements are carried out using Shimadzu UV-Visible computerized double-beam spectrophotometer UV-160.In all measurements, 1-cm matched cells are used.The pH measurements are carried out using (HANNA pH 211).

Reagents
All chemicals used are of highest purity available.

Working bismuth solution(100 µg/ml).
It is prepared by diluting 10 ml of the stock solution to 100ml with distilled water in a volumetric flask.

Working bismuth solution(10 µg/ml).
It is prepared by diluting 10 ml of the working solution (100 µg/ml) to 100 ml withdistilled water in a volumetric flask.

Bromopyrogallol Red (BPR) reagent solution (1×10 -3 M).
It is prepared by dissolving 0.055 g of BPR (BDH) in distilled water and the solution is diluted to 100 ml in a volumetric flask.The solution is transferred to a brown bottle where it remains stable for a least one week.

Buffer solution pH 3
It is prepared by mixing 50 ml of 0.1 M glycine solution with 5.7 ml of 0.2 M HCl solution and diluting the volume to 100 ml with distilled water in a volumetric flask, (Perrin and Dempsey, 1974).

Cetylpyridinium chloride (CPC) solution (2×10 -3 M)
It is prepared by dissolving 0.179 g of CPC monohydrate (Fluka) in distilled water and the solution is diluted to 250 ml in a volumetric flask.

Triton X-100 solution (4%)
This solution is prepared by diluting 4 ml of Triton X-100 (Scharlau) with hot distilled water and the solution is diluted to 100 ml in a volumetric flask.

Mixed complexing solution
This solution is prepared by dissolving 0.3 g of tartaric acid (BDH) with 0.042g of sodium fluoride) (BDH) in about 80 ml distilled water.The pH of the resulting mixture is adjusted to 3 with 0.1 M HNO 3 solution and the volume is then completed to 100 ml with distilled water.

Ascorbic acid solution (0.01M)
It is prepared by dissolving 0.1761 g of ascorbic acid (BDH) in distilled water.The pH of the solution is adjusted to 3 with nitric acid solution and the volume is completed to 100 ml with distilled water.

Veterinary preparation solution (1000 µg/ml )
The content of 6 sachets of Diaclean containing 2000 mg bismuth subnitrate was weighed.A quantity of powder containing 0.1 mg Bi 3+ is then dissolved in 5 ml of 5 M nitric acid and sufficient amount of distilled water.The solution is shaken thoroughly and then filtered.The filtrate was made up to 100 ml with distilled water in a volumetric flask.The tested solution is prepared by an appropriate dilution.

RESULTS AND DISCUSSION
Preliminary studies of the reaction of bismuth (III) with BPR reagent indicate that the reaction proceed immediately after mixing.The violet coloured complex showed an absorption maximum at 597nm in contrast to the reagent blank which shows maximum absorption at 522nm.

Study of the optimum reaction conditions
The effects of various parameters on the absorption intensity of the coloured complex are studied and the reaction conditions are optimised.

Effect of pH
The effect of pH on the colour intensity of the complex is studied with solutions containing 100 µg of Bismuth (III) and various volumes of 0.01M HNO 3 and NaOH solutions and 2 ml of (1×10 -3 M) BPR reagent and the volume is completed to 25ml with distilled water.The absorbance of each coloured solution against its corresponding blank solution and the final pH of the reaction mixtures are both measured.The results indicate that the absorbance is pH dependent, and the maximum absorbance occurred at pH 3 with maximum wavelength 602 nm, therefore it is selected for subsequent experiments.

Effect of buffers
A series of various buffer solutions of pH 3 are prepared and their effects on the absorbance, λ max and the pH of the final reaction mixture are examined.Seven buffer solutions of pH 3 such as tartaric acid-NaOH (B 1 ), citric acid-NaOH (B 2 ), khphalate -HCl (B 3 ), glycine -HCl (B 4 ) citric acid -sod.citrate (B 5 ),acetic acid -sodium acetate (B 6 ) and succinic acid -NaOH (B 7 ), (Perrin and Dempsey, 1974) are tested for the purpose.The results are given in Table (1).

Final pH of the reaction mixture
It can be noticed in Table (1) that buffer solution (B 4 ) is only useful from the analytical point of view.The other buffers show an un-encouraging results.For the subsequent work, a 2 ml of buffer (B 4 ) solution has been chosen because it gives the highest sensitivity and good colour contrast.

Effect of surfactants
The effect of the presence of cationic (Cetylpyridinium chloride, CPC, Cetyltrimethylammonium bromide, CTAB), anionic (Sodium dodecyl sulphate, SDS) and non-ionic (Iso-octylphenoxypolyethoxy ethanol, Triton X-100) surfactants on the colour intensity of the complex is examined with different orders of addition.The results are shown in Table (2).The results in Table (2) indicate that CPC solution causes bathochromic shift and increasing in the intensity of absorbance of formed complex with order of addition No.(II).Therefore the effect of different volumes of higher concentration of CPC (2×10 -3 M) on the intensity of coloured complex has been studied [Table (3)].The results obtained in Table (3) indicate that the addition of 4 ml of CPC with order No. (II) [Table (2)] gives maximum absorbance and the lowest blank value, so it has been used in the subsequent experiments.

Effect of mixed surfactants and temperature
It is found that the presence of 4% Triton X-100 solution with 2 × 10 -3 M CPC in the reaction mixture, an increase in the absorbance of the coloured complex is observed obviously especially when the reaction mixture is heated.The effect of temperature on the absorbance of the coloured complex is shown in Table (4).From the results in Table 4, it is observed that maximum absorbance and a good colour contrast are obtained at 50C˚.The effect of heating time on the absorbance of the coloured complex is then studied and the experimental results showed that a heating time of 5-7 min, was enough for the completion of the reaction.

Effect of the amount of Triton X-100
The effect of different volumes of 4%Triton X-100 on the intensity of the formed complex has next been studied.The experimental results showed that 1 ml of 4%Triton X-100 was optimum.

Effect of reagent amount
Different volumes of 1×10 -3 M BPR solution are added to 10-125 µg of bismuth while other conditions being kept constant.A 2 ml of the reagent solution has been found to be optimum volume since the linearity (correlation coefficient) is good and the sensitivity of the colour reaction is fair.

Effect of masking agents
The presence of tartaric acid, NaF and ascorbic acid in the reaction mixture is essential because they increase the selectivity of the method by masking cations from their reactions with BPR.Other complexons such as EDTA, EGTA and NTA decrease the absorbance owing to their complexons action with bismuth.Therefore, a composite masking solution has been prepared containing 0.01M NaF and 0.02M tartaric acid.Since the solution of ascorbic acid is unstable, it is added separately.The effect of composite complexing solution (with different volumes of 0.01M ascorbic acid) on the absorbance of coloured complex is examined.The results are given in Table ( 5) The results indicate that 2 ml of both mixed complexing solution and ascorbic acid are desired for subsequent experiments because of the highest sensitivity.

Effect of order addition
The orders of addition of solution are examined and the results are shown in Table ( 6) Table 6 : Effect of order addition.
From the results above, order (II) has been used for a subsequent experiment, due to the highest sensitivity.

Effect of time
A study of the time effect on the absorbance of the final reaction mixture shows that the maximum absorbance is obtained after 20 minutes and remains stable for at least 1 hour.

Final absorption spectra
Under the above established conditions, absorption spectra of a blue complex of bismuth-BPR-CPC-Triton X-100 and of its reagent blank are recorded and are shown in Fig .(1).The coloured complex exhibits maximum absorbance at 644 nm in contrast with the reagent blank which shows maximum absorbance at 533 nm.

Recommended procedure and calibration Graph
Sample aliquots containing 3-250 µg of bismuth solution are placed into 25-ml volumetric flasks.To each bismuth solution, 2ml of mixed complex solution (0.01M NaF and 0.02M tartaric acid), 2ml of 0.01M ascorbic acid, 2ml of buffer pH 3, 4ml of 2×10 -3 M CPC, 1ml of 4% Triton X-100, and 2 ml of 1×10 -3 M BPR are added.The solution is mixed and diluted to the mark with distilled water and heated to 50 C˚ for about 5 min in water bath.After about 10 min, the absorbance of each coloured solution at 644 nm is measured against the reagent blank, prepared in the same manner but without bismuth.Beer , s law is obeyed within the range (3-150) µg of Bi (III) in a final volume of 25 ml (i.e., 0.12-6 ppm) [Fig. (2)].The molar absorptivity and Sandell's sensitivity are 3.3×10 4 l.mol -1 .cm - and 0.006 µg.cm -2 , respectively.Fig. 2: Calibration graph for Bi 3+ determination using the proposed method.

Accuracy and precision
To check the accuracy and precision of the cribration graph, bismuth (III) is determined at three different concentrations.The results shown in Table (7) indicate that the method is satisfactory.

Nature of the complex
The stoichiometry of the complex has been studied by both Job's method of continuous variations and mole-ratio method (Delevie, 1997).The results reveal that the combination ratio of bismuth (III) with BPR is (1:2).Both methods are used to study the proportion of CPC in the complex and the results obtained do not give the expected proportion, as the colour of the solution changes with the addition of different amounts of CPC.The average of the stability constant of the coloured complex is found to be (4.3×10 11) M -2 (Hargis, 1988).The empirical formula of the chelate can be written as follows.

Effect of Interference
The influence of diverse ions on the determination of bismuth is examined under the conditions of standard procedure.The diverse ions are added, individually, to solutions containing 100 µg of bismuth.The results are summarised in Table ( 8), from which it can be concluded that the method seems to be selective except towards Al 3+ , Fe 2+ , Mn 2 + , Ni 2+ and Zr 4+ ions.Also, the effect of foreign compounds that may be present in dosage form were studied by adding different amounts of foreign substances to 100 µg Bi 3+ /25ml.It was observed that the studied foreign species did not interfere in the present method Table (9).

Application of the method Determination of Bi (III) in waters
The proposed method has been applied to the determination of bismuth (III) in various water samples, the results are shown in Table (10).The results listed in Table (10) show that the method is successful for determining Bi (III) in the above water sample.

Determination of Bi (III) in a veterinary preparation
The proposed method has also been applied to the determination of Bi (III) in veterinary preparation.The results are listed in Table (11).The above results reveal that the method is suitable for determining bismuth in the above sample with satisfactory recovery.Both the present method and the literature method (Marczenko, 2000) have been applied at the same time to t-test (Christian, 2004) and the value compared with the statistical tables for eight degrees of freedom at 95% validation level.The results in Table (12) show that there is no real difference between the two methods.

Comparison of methods
Table (13) shows the comparison between the analytical variables of the present method with those of other methods for bismuth determination.Comparatively, the present method is simple, sensitive and applicable to waters and veterinary preparation.

CONCLUSION
The proposed spectrophotometric method is simple, sensitive and low cost, it does not involve solvent extraction steps and gives precise and accurate results.The method has been applied successfully to the determination of bismuth in water samples and a veterinary preparation.

Table 4 : Effect of temperature.
*Using 1 ml of Triton X-100

Table 7 : Accuracy and precision.
*Average of five determinations.

Table 13 : Comparison of method.
*Involves flash point extraction.**Involves extraction in chloroform.